JPH0690737A - Centrifugal separation apparatus for biocell and cell cultivation method - Google Patents

Abstract

PURPOSE:To provide a centrifugal separation apparatus capable of centrifugally separating the cells from a culture liquid in the suspension irrigation culture of biocells, especially animal cells and suspending the separated cells in a medium in high efficiency without damaging the cells and to provide a high-speed irrigation culture system containing the apparatus as an element. CONSTITUTION:The centrifugal separator has a horizontally rotatable bowl-type rotary vessel 1 containing a finned axial pipe 2. The centrifugal separator (1) has a plurality of blade pieces 3 extending from the axial pipe in the circumferential direction to form a narrow gap between the outer edge of the piece and the inner wall of the rotary vessel, (2) has a window 15 on a surface of the blade and a door 16 closing the window 15 and freely openable in the same rotational direction, (3) forms a clutch 4 with the lower end of the axial pipe 2 and the bottom of the vessel 1 to disconnect in the rotational direction and connect in the reverse rotation direction and (4) forms a freely rotatable sliding face at the upper end of the axial pipe 2 and a stationary pipe 5b above the axial pipe. The lowering of the precipitation efficiency of cells caused by the flow of the liquid in the acceleration of the rotation of the centrifugal separator can be prevented to enable the precipitation in high efficiency. Furthermore, the overflow of the liquid from the vessel can be prevented in the suspension of the separated cell layer in the medium to enable the effective suspension of the cells in the medium.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to separating and recovering cells from a liquid containing biological cells and suspending them in a fresh medium under suitable conditions without damaging biological cells and invading microorganisms. TECHNICAL FIELD The present invention relates to a biological cell centrifuge, and a cell culture method and system using the centrifuge.

[0002]

2. Description of the Related Art In recent years, plant cells produced by cell fusion including animal cells have been perfused to collect useful components from the culture solution. Perfusion culture is performed by repeating the process of separating cells from fresh medium in the culture system by filtration or centrifugation and resuspending in fresh medium. Among them, centrifugation has a high separation rate, and thus a higher perfusion rate can be obtained as compared with filtration. For this reason, centrifugation has recently attracted attention as a cell separation element for perfusion culture.

Up to now, a continuous treatment system has been mainly used for perfusion culture of biological cells. That is, the culture solution is continuously supplied into a rotating centrifuge rotating container,
It is a cell concentrate and a supernatant which are continuously separated and taken out. Examples of continuous processing centrifugal separators and separation methods are as follows: 1) Bernard Jeu Van Wee Pullman et al., US Pat. No. 4,939,087 (publication date: July 3, 1990).
2) Bernard Jou Van Wi Pullman et al., Biotechnology and Bioengineering, Vol. 38, pp. 1190 to 1202 (1991). In these continuous treatment methods, the cells in the culture medium come into contact with the inner surface of the rotating container that rotates at high speed, and the cells are easily damaged. In particular, fragile animal cells among biological cells are easily damaged, and it is difficult to obtain a high survival rate of 90% or more.

In order to make up for the drawbacks of such a continuous treatment system, the culture solution is supplied in a state where the rotary container is allowed to stand, and after the completion of the supply, the rotary container is rotated to separate and resuspend in a fresh medium. There is a batch treatment method, and as an improved version of the method, the present inventors arranged a wing piece (a partition wall provided inside the rotary container) around a shaft tube having a hole for inflow and outflow of culture solution in the rotary container. Then, at the time of centrifugation, the rotating container and the blades are connected and rotated in an integrated state to perform centrifugal separation,
An improved centrifuge device in which cells are suspended by agitating a culture solution by separating the rotating container and the wing piece with the culture medium after the supernatant is discharged and rotating them at different angular velocities, and the same A method for separating cells using the method has already been proposed (see Japanese Patent Laid-Open No. 2-131154).

[0005]

DISCLOSURE OF THE INVENTION The inventors have conducted a number of experiments using the above centrifuge and, through the experiments,
When the difference between the cross-sectional area of the winglet and the cross-sectional area of the rotary container is large, that is, the wing surface area of the winglet is smaller than the cross-sectional area of the rotary container, the inner surface of the rotary container and the winglet wing tip (ie If the gap between the blade and the edge of the blade that faces the inner wall of the rotary container is large, when the rotation of the rotary container is accelerated, the liquid in the rotary container flows due to the inertial force due to the existence of the gap, and the sedimentation path of the cells is formed. It was found that it took a long time to prolong and to completely settle the cells. The longer the time required for the separation, the lower the efficiency. It is possible to prevent the flow of the inner liquid due to so-called co-rotation to some extent by gradually accelerating the rotating container, but this also leads to a decrease in processing efficiency. Therefore, the work was performed by filling the gap between the inner wall of the rotating container and the blade tip of the blade to increase the shielding effect. As a result, the flow of the liquid was suppressed and the cells could be settled and separated on the inner wall of the rotating container in a short time.

However, after the supernatant is discharged, fresh medium is supplied into the rotary container, and only the rotary container is rotated with the wing pieces fixed to suspend the cells that have settled and separated on the inner wall of the rotary container in the medium. Then, it was found that the liquid overflowed from the upper edge of the rotating container even when the rotating container was rotated at a low speed. In order to prevent overflow, it is necessary to extremely reduce the amount of the liquid to be poured into the rotating container, and with that, the amount of the culture liquid to be subjected to centrifugation must be significantly reduced. It turns out that efficiency cannot be increased.

Therefore, the inventors of the present invention were able to effectively prevent the flow of the liquid in the rotary container during centrifugation and, even when the amount of the internal liquid filled was sufficiently large during cell suspension, the liquid flowed from the upper edge of the rotary container. As a result of extensive studies on a method and an apparatus for preventing the overflow of water, the present invention has been achieved.

[0008]

The first feature of the present invention is to:
A rotary container having an upper opening for containing the culture solution, a shaft tube having a through hole for introducing and discharging the culture solution, a plurality of blades radially fixed to the shaft tube and positioned in the rotary container, and In a centrifuge for biological cells, which has a drive means for selectively causing relative rotation and synchronized rotation between the rotary container and the blade, in the relative rotation and synchronized rotation of the blade. It is to have means for changing the effective area acting on the culture solution depending on time.

As means for changing the effective area,
An opening is formed in a part of the wing piece and a door capable of opening and closing the opening is attached to the opening, or a part of the wing piece is folded only in one direction at a position close to the inner wall of the rotary container. Making it bendable is a preferred embodiment. Further, a one-way clutch is interposed between the bottom portion of the rotary container and the shaft tube as a part of a driving unit that selectively causes the rotary container and the blade to selectively rotate in a relative and synchronized manner. In addition, it is a preferable embodiment that the magnetic force transmission mechanism is constituted by the magnet arranged inside the rotary container and the permanent magnet arranged outside the rotary container. Then, the relative rotation of the rotary container and the wing piece is caused by the one-way clutch being in a disengaged state and the rotary container rotating and the wing piece being substantially fixed, and
Synchronous rotation of the rotary container and the blades is generated by turning on the one-way clutch.

The tip end of the shaft tube is connected to a different fixed shaft tube which is located at a higher position and has a through hole for introducing and discharging the culture solution, through a clutch mechanism having a sliding contact surface. The system configuration described later becomes easier. Also,
By further providing another liquid supply pipe having a nozzle for supplying the culture solution toward the inner wall of the rotary container at the tip, the suspension of cells becomes smoother.

The position and shape of the opening provided in the wing piece are not particularly limited. For example, it may be a notch provided downward from the upper edge of the wing, or in the direction of the rotation axis from the side edge of the wing, or from the lower edge of the wing to the upper portion, or a hole may be opened in the wing surface. May be. It will be easily understood that the door may be any one that can sufficiently block the above-mentioned opening and can be opened and closed in a specific direction. A conventionally known method such as a hinge is the simplest method for opening and closing.

It is preferable that the gap between the inner wall of the rotary container and the tip edge of the blade is as small as possible, but when the blade is fixed and the rotating container is rotated, the rotary container is freely rotatable without sliding contact with each other. The smallest possible gap, preferably less than 2 mm, is used. In addition, in the case of culturing animal cells, the gap during suspension can be reached even if high density culture of 1 × 10 ⁸ cells / ml is reached.
Since the thickness of the cell layer deposited on the inner wall of the rotary container is 5 mm or less, 5 mm is sufficient. If the gap is 10 mm or more, the mixing efficiency of the liquid as the baffle is lowered, and it takes time to suspend the cell layer, particularly the cell layer of a kind having high adhesiveness. Therefore, a gap of 5-10 mm is preferred for effective suspension.

In another aspect of the present invention, as a means for changing the effective area of the wing blade depending on the relative rotation and the synchronized rotation, the end of the wing blade adjacent to the inner wall of the rotating container is used. It is also possible to integrally form an elastic member on the edge and extend the elastic member by centrifugal force. In this aspect, the tip of the elastic member can be brought into close contact with the inner wall surface of the rotating container during centrifugation.
Thereby, the flow of the liquid in the rotary container is substantially blocked, and the cells are effectively deposited on the inner wall of the rotary container, and when the cells are suspended, the rotation of the rotary container is not only smoothed,
The appropriate gap as described above is formed between the inner wall surface of the rotating rotary container and the peripheral edge of the fixed blade so that the cells are not damaged and the blade has the effect of a baffle.

The present invention further connects the above-mentioned centrifugal separator for biological cells with a culture tank or a cell-containing liquid storage tank, a centrifugal supernatant liquid storage tank, and a liquid culture medium storage tank by piping, and each element on the piping by a process sequencer. Also disclosed is a biological cell culture system characterized in that adjustment is performed by operating a pump and opening / closing a valve, and further, a method for culturing cells by a process in which at least the following unit steps are arranged in the following order: Is also disclosed.

1) A step of quantitatively supplying a cell-containing liquid from the outside of the rotary container into the rotary container. 2) A step of performing centrifugal separation by the synchronized rotation of the rotary container and the blade. 3) A step of stopping both the rotation of the rotary container and the rotation of the winglet. 4) A step of discharging the centrifugal supernatant to the outside of the rotary container through the through hole for introducing and discharging the culture solution formed in the shaft tube.

5) While rotating the rotary container and the wing piece relatively, the rotary container is rotated through a through hole for introducing and discharging the culture liquid formed in a liquid supply pipe or a shaft tube having a nozzle tip for supplying the culture liquid toward the inner wall of the rotary container. A step of quantitatively supplying a cell suspension medium solution into the container. 6) A step of causing relative rotation between the rotating container and the blade to suspend the cells in a medium. 7) A step of discharging the cell suspension to the outside of the rotary container through the through hole for introducing and discharging the culture solution formed in the shaft tube.

8) Waiting step. By repeating the above steps 1 to 7 at least once or more, more reliable cell separation can be performed. The present invention further discloses a method for separating cells, characterized by performing steam sterilization comprising the following steps prior to the first step.

1) A step of introducing steam into the rotary container and discharging air from the rotary container. 2) A process of maintaining the state at 120 ° C or higher for 10 minutes or longer. 3) The step of closing the pipe coming out of the rotating container. 4) Step of cooling. 5) A step of introducing sterile air into the rotating container and equilibrating the atmospheric pressure inside the rotating container with the external pressure.

6) A step of discharging the drain inside the rotary container to the outside of the rotary container. By the way, in order to provide the centrifuge according to the present invention for culturing animal cells, it is essential to have a means for separating and resuspending the cells in a substantially sterile environment, since various bacteria enter under an open condition. Become. For this purpose, the rotary container is driven by a rotary container and its accessories are arranged inside a container consisting of steam pressure resistant walls, and the rotary container is rotated by a permanent magnet at the bottom of the rotary container and It is a preferred embodiment to use a magnetic force transmission mechanism using a permanent magnet under the bottom wall of the container.

[0020]

When the centrifugal separator for biological cells according to the present invention is used, after sterilizing the inside of the rotary container with steam as required, the rotor chamber is cooled until the pressure reaches atmospheric pressure, and then sterile air is passed through the filter. It is introduced into the rotor chamber to keep the temperature in the rotor chamber at the culture temperature. After draining the drain in the rotor and in the rotor chamber to the outside of the system, a predetermined amount of the cell-containing liquid is supplied from the culture tank outside the rotary container or the cell-containing liquid storage tank into the rotary container. Then, the drive mechanism is operated to put the clutch in the engaged state, and the rotary container and the blade are synchronously rotated to perform centrifugal separation.
The inner wall of the rotary container and the tip edge of the winglet are in close contact with each other due to the centrifugal force generated by the synchronous rotation, or preferably set to 2 mm or less and the opening formed in a part of the winglet is closed. Therefore, the effective area of the winglets acting on the culture solution is increased. In other words, since the work can be performed by filling the substantial gap between the inner wall of the rotary container and the blade tip of the blade to enhance the shielding effect, the flow of the liquid is suppressed and the cells can be stored in the rotary container in a short time. It is possible to settle and separate on the inner wall.

After the rotation for a predetermined time, the rotation of the rotary container and the blades are stopped together, and the centrifugal supernatant is transferred to the centrifugal supernatant reservoir outside the rotary container through the through hole for introducing and discharging the culture solution formed in the shaft tube. Discharge. Next, a predetermined amount of the cell suspension medium liquid is supplied from the liquid medium storage tank into the rotary container through the culture liquid introducing / discharging through hole formed in the shaft tube or via the liquid supply pipe provided elsewhere.

The drive mechanism is operated again with the medium filled in the rotary container. At this time, by disengaging the clutch, only the rotating container rotates and the wing pieces stop. As a result, in the embodiment in which the elastic member is integrally formed on the edge of the wing piece, the elastic member returns to its original posture, and cells are formed between the inner wall surface of the rotating container and the peripheral edge of the fixed wing piece. The above-mentioned appropriate gap is formed so that the winglet can exert the effect as a baffle without being damaged. Further, in a mode in which an opening is formed in a part of the wing piece and a door is attached to the opening, or in a mode in which a part of the wing piece is bendable in only one direction at a position close to the inner wall of the rotating container. In the thing, by opening the door and opening the opening, or by bending a part of the wing piece to form the opening,
The wing pieces fulfill the function of an obstruction plate, and the cells are suspended in the medium and at the same time, the culture solution can be moved to some extent in the rotary container, so that the liquid is prevented from overflowing from the upper edge of the rotary container. .

After the suspension operation for a predetermined time, the rotation of the rotary container is stopped, and the cell suspension is discharged into the culture tank outside the rotary container through the through hole for introducing and discharging the culture solution formed in the shaft tube. As described above, in the apparatus for culturing cells and the method for culturing cells of a living cell according to the present invention, it is possible to effectively prevent the flow of the liquid in the rotating container during centrifugation, and to adjust the filling amount of the internal liquid during cell suspension. Even if it is sufficiently large, the liquid does not overflow from the upper edge of the rotary container, so that efficient perfusion culture can be performed.

[0024]

EXAMPLES Next, examples will be shown and described in more detail. [Embodiment 1] An embodiment of a centrifugal separator for living cells according to the present invention is shown in FIGS. A shaft tube 2 with blades 3 is arranged in a bowl-shaped (bowl-shaped) rotary container 1 having a trapezoidal cross section. The shaft tube 2 has an axial through hole 2a at the center thereof, and the lowest part thereof forms a gear clutch 4 with the center portion of the bottom of the rotary container. As shown in FIG. 2, the gear clutch 4 is in an engaged state, that is, when the gear clutch portion 4a at the lower portion of the shaft tube 2 is in contact with the gear clutch portion 4b at the bottom portion of the rotary container, and is disconnected when the shaft tube 2 is raised. It is like this. The gear clutch portion 4b at the bottom of the rotary container has a vertical hole 44 and a lateral hole 43 communicating with the vertical hole 44 at the center thereof, and when it engages with the other gear clutch portion 4a, the through hole formed in the shaft tube 2 is formed. Hole 2a, Vertical hole 44, Horizontal hole 43
And communicate with each other to form a passage for liquid in and out.

The wing piece 3 has a window 15 cut out downward from the upper edge thereof, and a door 16 having an area sufficient to shield the window 15 is attached to the window 15 by a hinge 17. The rotary container 1 has a rotary shaft 45 located on the bottom rear surface thereof on the same axis as the shaft tube 2, and a permanent magnet plate 21a is provided at the bottom of the rotary shaft 45. A rotary shaft 45 of the rotary container is rotatably supported in a vertical direction by a bearing bearing 19 supported by a support arm 20. The support arm 20 is mounted on a container bottom plate 34 that forms the bottom of a container 46 that is a pressure resistant wall that houses the rotary container 1. The permanent magnet 21a faces the permanent magnet 21b which is in contact with the motor 35 on the pedestal 36 with the container bottom plate 34 sandwiched between them. It is transmitted to the rotary container 1.

The upper end of the shaft tube 2 becomes a curved large-diameter convex portion 5a, which is slidably fitted to the slide support cylinder 12 fixed to the support arm 14 standing on the container bottom plate 34 and the upper end is opened. It is located in the outer casing 6 with its large diameter portion in contact with the bottom portion of the outer casing 6. An inner casing 7 whose lower part is opened is fitted in the outer casing 6, and the inner casing 7 has a second shaft tube having a recess 5b at the lower end which is in sliding contact with the projection 5a at the upper end of the shaft tube 2. 2b
Is inserted in a state in which the concave portion 5b at the lower end thereof is in sliding contact with the convex portion 5a at the upper end of the shaft tube 2. The recess 5b is constantly pressed downward by a spring 8 inserted between the upper portion of the inner casing 7 and the recess 5b, and forms a ball joint 5 that maintains airtightness with the protrusion 5a.

Similarly to the first shaft tube 2, the second shaft tube 2b also has an axial through hole in the center thereof. The upper end of the second shaft tube 2b is led to the outside of the rotary container 46 by the pipe 26 through the flexible tube 9 made of a stretchable material. The pipe 26 is fixed by a packing 24 supported by a container top plate 30 and a screw lid 25. The pipe 26 serves as a medium supply and a centrifugal supernatant discharge as described later.

Support arm 14 adjacent to slide support tube 12
A bellows 10 is provided on d, and the upper end of the bellows 10 and the upper end of the outer casing 6 have vertical arms 41, 41 ',
The horizontal arm 11 and the fulcrum 47 are connected by a link mechanism. The inside of the bellows 10 is connected to a pressurized air source 39 via a pipe 13. Therefore, by introducing the pressurized air into the bellows 10 through the pipe 13, the arms 41 and 41 'are raised around the fulcrum 47, and the outer casing 6 is pulled up along the slide support cylinder 12, and with it The curved large-diameter convex portion 5a at the upper end of the shaft tube 2 also rises.
That is, by supplying pressurized air to the bellows 10, the flexible pipe 9 is deflected, and the winglets 3 and the shaft pipe 2 in the rotary container 1 are lifted while maintaining the airtightness of the ball joint portion 5 to lift the clutch 4. Can be cut to disconnect the connection with the rotary container 1.

In the container top plate 30, an introducing pipe 47 for introducing the steam 27 for sterilizing the inside of the container, a microbial filter 29 for balancing with the outside air 28 after completion of steam heating sterilization, and an outside air introducing pipe 48 are similarly provided. The packing 24 and the screw lid 25 are fixed and arranged. The container body 31a is provided with a jacket 31b having a constant temperature water inlet 32 and an outlet 33 so that it can be kept warm by constant temperature water, and the container body 31a and the top plate 30 and the bottom plate 34 are sealed by a packing 42. Also,
The bottom plate 34 has a drain discharge pipe for discharging the drain 37.
38 is provided via packing 24.

Further, by further providing another different liquid supply pipe 22 having a nozzle 23 for supplying the culture solution toward the inner wall of the rotary container 1, the suspension of the cells becomes smoother.
Next, the operation of the above centrifuge for biological cells will be described based on an example in which the centrifuge is incorporated into an actual biological cell culture system for culturing. [Second Embodiment] FIG. 8 is a centrifuge shown in the first embodiment.
66 shows a culture system in which the culture tank 56, the culture medium storage tank 63, the centrifugal supernatant storage tank 49, and the steam generator 45 are connected by pipes.
By-pass pumps and transfer pumps were arranged in the middle of each element and piping, and each valve and each pump were electrically connected to a sequencer 46 that controls the on / off sequence.

The culture tank 56 has a volume of 2 L of the infusing liquid, and the aerated enzyme-containing gas 66 can be aerated through the filter 51 under agitation to supply the dissolved oxygen. The culture medium storage tank 63 and the centrifugal supernatant storage tank 49 are each provided with an air filter 51 for removing the invasion of microorganisms from the outside atmosphere. Prior to culturing, steam is sequentially supplied to each tank and each piping system of the above system, and sterilized by heating at 125 ° C for 30 minutes or more. Steam supply for steam generator
From 45 through pipe 55, valve 47, pipe 55 into culture tank 56,
Further, the centrifugal separator 66 and the centrifugal supernatant storage tank 49 are supplied via a pipe 55, a valve 47, and a pipe (not shown), respectively. The drains of the steam drain are pipes 67, 37, respectively.
It is discharged by 66 and 68. The drain remaining in the rotary container 1 of the centrifuge 66 is the hole 2a formed in the tube body 2 described above.
Through pipes 26, 66, valve 52, and pump 48 into a supernatant storage tank
Discharged to 49.

After steam sterilization, bovine serum was added to the medium storage tank 63.
Dulbecco's modified Eagle MEM medium 62 supplemented with 10% was filled. The medium 62 was supplied into the culture tank 56 through the pipe 65, the pump 64, the flow path switching valve 70, and the pipe 55, and the temperature was kept at 37 ° C. Mouse-mouse hybridoma cell line STK-1 at 1 x 10
Inoculate to a concentration of ⁶ cells / ml from the pipe 61, 37 ℃, 30rpm,
Culture was started under the conditions of a dissolved oxygen concentration of 2 ± 0.2 ppm and a pH of 7 ± 0.1. The liquid phase gas phase portion of the culture tank 56 has an antifoaming net 57 impregnated with organosilicon as an antifoaming agent,
When aeration is passed through the culture medium 60, it contacts the foam layer generated on the liquid surface to defoam it, and the exhaust gas is drained by the drain remover 59, sterilization filter.
It was exhausted to the outside of the system via 51.

One-third of the amount of the infused solution 12 hours after the inoculation
0.67 L of the culture solution 60 corresponding to the amount was supplied into the centrifuge rotary container 1 through the pipe 22, the pump 54, the valve 53, the pipe 26 and the hole 2a of the winged shaft tube 2. Next, in a state where the pressurized air 39 is not introduced into the bellows 10, that is, the gear clutch portion 4a of the winged piece shaft tube 2 and the gear clutch portion 4 of the rotary container 1.
In the state where b is engaged with the shaft tube with winglet 2 and the rotary container 1, the two are synchronously rotated clockwise at 2000 rpm for 2 minutes by the motor 35 to deposit cells in the culture solution on the inner wall of the rotary container 1. Let The gap between the blade edge and the inner wall of the rotating container during centrifugation was set to 3 mm or less. It took about 0.5 minutes until the rotating container 1 stopped after rotating for 2 minutes. After the rotation was stopped, the supernatant 50 in the rotary container 1 was stored in the supernatant storage tank 49 through the hole 2a of the shaft tube 2 and the pipes 26 and 66 with the valve 53 closed and the valve 52 opened.

Next, pressurized air 39 is supplied to the bellows 10 via the pipe 13 to lift the shaft tube 2 with blades, thereby disengaging the gear clutch 4 and connecting the shaft tube 2 with blades and the rotary container 1. Separated. By the motor 35, while rotating only the rotating container 1 in the same direction as during centrifugal separation, that is, clockwise at a low speed of 30 rpm, 0.67 L of the medium 62 in the medium storage tank 63,
The pump 64 was operated to inject it through the pipes 65 and 23 onto the inner wall of the rotating rotating container 1. During this time, the inner solution in the rotary container 1 opens in the clockwise direction by the hinge 17 on the door 16 of the wing piece 3, the content liquid flows through the window 15, and no overflow is observed from the upper edge of the rotary container 1. It was confirmed that the cells were uniformly suspended in the medium by spinning for 5 minutes.

The pressurized air in the bellows 10 is extracted from the pipe 13, the shaft tube 2 with blades is lowered, and the rotary container 1 and the clutch 4 are connected to each other.
Pump through the wing piece shaft tube 2, piping 26, valve 53, piping 23
It was returned to the culture tank 56 by 54. Do the above operations from 3 to 1 day
Perfusion culture was continued by performing 15 cycles. By adjusting the waiting time between cycles, the frequency of cycles, ie the perfusion rate, can be adjusted at will. The culture results are shown in Table 1.

As shown in Table 1, there was no difference in the cell concentration between the culture solution and the cell suspension obtained by suspending the cells in the medium after centrifugation, and no loss of the solution was observed. This result shows that when suspending cells, the cells can be uniformly dispersed without overflowing the cell suspension from the upper end of the rotating container. It is also clear that the viability of both the culture and the cell suspension are the same, and that the cells are not damaged, including both the centrifugation and suspension steps. Furthermore, during the culture period of 30 days, the medium exchange amount can be stepped up to 1 to 5 vol-culture tank overhanging amount / day without detecting microorganisms in the culture medium, that is, without allowing the invasion of microorganisms from outside the system. As a result, a high cell concentration of 4 × 10 ⁷ cells / ml was reached, and high-density perfusion culture was realized.

[0037]

[Table 1]

The above description of the apparatus for centrifuging biological cells and the method of culturing cells using the apparatus is merely one example, and there are many other variations. FIG. 5 shows another means for changing the effective area of the blade in the configuration of the centrifugal separator. In this example, a window-shaped opening 115 is formed in the blade surface of the blade 3, and the opening 115 is formed in the opening 115. To the door
The hinge 117 is pivotally attached so that it can be opened and closed only in one direction.

FIG. 6 shows still another means for changing the effective area of the blade, and in this example, the tip of the blade 3 (the portion in the rotary container 1 which is close to the inner wall of the rotary container) is arranged. The door 216 is separated and is hinged to the winglet body so that the door 216 can be opened and closed only in one direction by a hinge 233. The winglet structure shown in FIGS. 5 and 6 is also shown in FIG.
It will be readily understood that the same function as the winglet shown in FIGS.

FIG. 7 shows yet another means of varying the effective area of the winglet, which in this example is shown in FIGS.
As in the case of the winglet formed on the winglet shown in Fig. 5, from the window 15 cut out from the upper edge of the winglet 3 and the door 16 having an area sufficient to shield the window 15 attached by the hinge 17. In addition to the above means, an elastic material such as a foamed material such as polypropylene, heat resistant synthetic rubber, polypropylene resin or the like is provided at the tip (the portion in the rotary container 1 which is close to the inner wall of the rotary container) and the lower end of the wing piece 3. Is provided. And
The size is such that a wider gap is formed between the tip edge of the winglet and the inner wall of the rotary container when stationary as compared with the case of the winglet structure described in FIGS. 1 to 6. There is.

In the wing piece having this structure, when the rotary container 1 and the wing piece 3 rotate synchronously, the elastic material 69 provided at the tip of the wing piece 3 comes into contact with the inner wall of the rotary container by the centrifugal force. To extend almost in the radial direction to increase its effective area.
Further, the elastic material provided on the lower side of the blade allows the gap between the lower side of the blade and the bottom of the rotary container to be substantially sealed when the clutch 4 is in the engaged state. Therefore, in this example, it is possible to effectively prevent the flow of the liquid in the rotary container during centrifugation more than in the other examples and to sufficiently increase the internal liquid filling amount during cell suspension. However, it becomes possible to prevent the liquid from overflowing from the upper edge of the rotary container.

Further, in FIG. 1, the shape of the rotary container has been described as a ball type (bowl shape) whose upper surface is narrower than the bottom surface. However, the shape of the rotary container is not limited to this shape and is cylindrical. Those having a slightly concave bottom surface and the like can also be effectively used, and it is particularly preferable that the shape of the wing pieces used at that time be the shape of the rotary container. It will also be understood that the number of wings is arbitrary and the angle of extension in the radial direction is not limited to 90 ° as shown. Further, as a modification of the winglet shown in FIG. 7, without using the means consisting of the window 15 and the door 16, an elastic body is formed at the tip and the lower end of the winglet 3, the winglet 3
Those in which an elastic body is formed only at the tip portion of can also be effectively used.

Of course, the configuration of the clutch 4 is also arbitrary, and it is effective to use a so-called one-way clutch (not shown) that transmits power for rotation in one direction but does not transmit power for rotation in the other direction. It will be understood that, in that case, the means for raising the shaft tube 2 including the bellows 10 shown in FIG. 1 is not necessarily required. Further, in the above description, an example in which the culture solution from the medium storage tank 63 is supplied into the rotary container 1 from the liquid supply pipe 22 having the nozzle 23 at the tip has been described. It may be carried out through a pipe 26 connected to the hole 2a formed in No. 2, or may be carried out through both the pipes 23 and 26.

Next, several comparative examples will be shown for the purpose of demonstrating the usefulness of the apparatus for centrifuging biological cells and the method for culturing cells according to the present invention. [Comparative Example 1] The same system and the same operating conditions and procedures except that the blades used in Examples 1 and 2 were replaced by flat blades having the same shape and size but no window and door. Culture experiments were carried out in.

The results are shown in Table 2. Although the cells were not damaged during suspension, the cells were overflowed from the upper edge of the rotating container and lost, so that the cell concentration in the culture tank could not be increased and the culture was stopped on the 5th day.

[0046]

[Table 2]

Comparative Example 2 The blade piece used in Comparative Example 1 is arranged along the outer peripheral edge so that the gap between the outer peripheral edge of the blade piece and the inner wall of the rotary container is 15 mm.
Example 1 except that it is replaced with a wing piece that has been cut so that
Culturing was carried out by the same system and method as those of Nos. 2 and 3. The results are shown in Table 3. During cell suspension, there is no overflow of cell suspension from the rotary container, and therefore there is no loss of liquid, but the cell layer deposited on the inner wall of the rotary container by centrifugation is not completely dispersed in the medium. Some cells are not returned to the culture tank with the cell clumps remaining in the rotating container. As a result, the cells remaining in the rotary container died, the cell concentration of the culture solution decreased, and the cell viability also decreased.

[0048]

[Table 3]

[0049]

According to the present invention, due to the above-mentioned winglet structure, the effective area of the winglet is increased by centrifugal separation force during rotation during centrifugal separation. Therefore, even if the liquid in the rotating container flows when the rotation is accelerated, the flow is limited to the space divided between the blades. Therefore, cells can be deposited on the inner wall surface of the rotating container by centrifugation for a short time.
After the centrifugation is completed, the supernatant is discharged, and the fresh medium is supplied, the blade is fixed and the rotating container is rotated at a low speed to open the door, etc., and the effective area of the blade for the culture solution. And the liquid in the rotating container flows and the cells attached to the inner wall of the rotating container can be efficiently suspended in the medium. At this time, as compared with the conventional example, even if the amount of liquid infused into the rotary container is increased, the liquid can be suspended without overflowing outside the rotary container.

As described above, as compared with the conventional example, a large amount of liquid can be processed in a short time, that is, the processing capacity can be increased.

[Brief description of drawings]

FIG. 1 is a sectional view showing an embodiment of a centrifugal separator according to the present invention.

FIG. 2 is a perspective view of an embodiment of a winglet of a centrifugal separator according to the present invention.

3 is a cross-sectional view of the blade and the rotary container of FIG.

FIG. 4 is a perspective view showing a clutch portion between the blade and the rotary container.

FIG. 5 is a perspective view showing another embodiment of the winglet according to the present invention.

FIG. 6 is a perspective view showing still another embodiment of the winglet according to the present invention.

FIG. 7 is a perspective view showing still another embodiment of the winglet according to the present invention.

FIG. 8 is a system diagram showing an example of a culture system according to the present invention.

[Explanation of symbols]

 1 Centrifugal Separation Rotating Container 2 Shaft Pipe 3 Wing Piece 4 Gear Clutch 9 Flexible Piping 10 Bellows 15 Window 16 Door 21a Magnet 31a Rotating Container Body 35 Motor 45 Steam Boiler 46 Process Sequence 49 Supernatant Tank 56 Culture Tank 63 Media Tank

Claims (15)

[Claims] 1. A rotary container having an open top for containing a culture solution, a shaft tube having a through hole for introducing and discharging the culture solution, a plurality of sheets radially fixed to the shaft tube and located in the rotary container. And a drive means for selectively causing relative rotation and synchronized rotation between the rotary container and the blade, the centrifugal separator for biological cells, wherein the blade is the relative A centrifuge for living cells, comprising means for changing an effective area acting on the culture solution between different rotations and synchronized rotations. 2. The centrifuge for biological cells according to claim 1, wherein the means for changing the effective area is an opening formed in a part of the wing piece and a door capable of opening and closing the opening. Separation device. 3. The means for changing the effective area is a bending means capable of bending a part of the blade piece only in one direction at a position close to the inner wall of the rotary container. The apparatus for centrifuging biological cells according to claim 1. 4. The biological cell according to claim 1, wherein the means for changing the effective area is an elastic member provided on the edge of the blade at a position close to the inner wall of the rotary container. Centrifugal separator. 5. The centrifuge for biological cells according to claim 1, wherein the driving means has a clutch means interposed between the bottom of the rotary container and the shaft tube. . 6. The centrifuge for biological cells according to claim 5, wherein the clutch means is a one-way clutch. 7. The relative rotation between the rotary container and the wing piece is configured such that the clutch means is in a disengaged state and the rotary container rotates to substantially fix the wing piece. The centrifuge for biological cells according to any one of claims 5 to 6, characterized in that. 8. The centrifuge for biological cells according to claim 5, wherein the rotation of the rotary container and the rotation of the wing piece in synchronization are generated when the one-way clutch is engaged. Separation device. 9. The upper end of the shaft tube is connected to another fixed shaft tube which is located further above and has a through hole for introducing and discharging a culture solution, through a joining mechanism having a sliding contact surface. The centrifuge for biological cells according to any one of claims 1 to 8, characterized in that 10. The magnetic drive mechanism according to claim 1, wherein the drive unit has a magnetic force transmission mechanism including a magnet arranged inside the rotary container and a permanent magnet arranged outside the rotary container. The centrifuge for biological cells described. 11. The biological cell according to claim 1, further comprising a different liquid supply pipe having a nozzle for supplying a culture solution toward an inner wall of the rotary container at a tip thereof. Centrifuge. 12. The apparatus for centrifuging biological cells according to any one of claims 1 to 11 is connected to a culture tank or a cell-containing liquid storage tank, a centrifugal supernatant liquid storage tank, and a liquid culture medium storage tank by piping to connect each element. A biological cell culture system characterized in that adjustment is performed by operating a pump on a pipe by a process sequencer and opening / closing a valve. 13. A method of culturing cells using the centrifuge according to any one of the first to eleventh aspects, by a process in which at least the following unit steps are arranged in the following order. 1) A step of quantitatively supplying the cell-containing liquid from the outside of the rotary container into the rotary container. 2) A step of performing centrifugal separation by the synchronized rotation of the rotary container and the blade. 3) A step of stopping both the rotation of the rotary container and the rotation of the winglet. 4) A step of discharging the centrifugal supernatant to the outside of the rotary container through the through hole for introducing and discharging the culture solution formed in the shaft tube. 5) Through the through hole for introducing and discharging the culture solution formed in the liquid supply pipe or the shaft tube having the nozzle tip for supplying the culture solution toward the inner wall of the rotation container while relatively rotating the rotation container and the blade.
A step of quantitatively supplying a cell suspension medium into a rotating container. 6) A step of causing relative rotation between the rotating container and the blade to suspend the cells in a medium. 7) A step of discharging the cell suspension to the outside of the rotary container through the through hole for introducing and discharging the culture solution formed in the shaft tube. 8) Stand-by process. 14. The first to seventh steps according to claim 13.
A method for separating cells, which comprises repeating the steps up to the step at least once or more. 15. The method for separating cells according to claim 14, wherein steam sterilization comprising the following steps is performed prior to the first step. 1) A step of introducing steam into the rotary container and discharging air in the rotary container. 2) A process of maintaining the state at 120 ° C or higher for 10 minutes or longer. 3) The step of closing the pipe coming out of the rotating container. 4) Step of cooling. 5) A step of introducing sterile air into the rotating container and equilibrating the atmospheric pressure inside the rotating container with the external pressure. 6) A step of discharging the drain inside the rotary container to the outside of the rotary container.